Several constitutional disorders in humans are characterized by an expanded region of trinucleotide repeats in a particular locus of an individual's genome. The two best-known disorders of this type are Fragile X syndrome and Huntington's disease. The number of trinucleotide repeats present at the locus of an individual's genome correlates with the severity of the disorder. Thus, various methods have been developed for determining the length of a trinucleotide repeat region of certain disorder-related genes. In Fragile X, the repeat motif is CGG. The established clinical method for diagnosis of Fragile X is the Southern blot, in which genomic DNA from an individual is digested by a restriction enzyme to excise the trinucleotide repeat region from the genomic DNA. This trinucleotide repeat region is then size-separated by electrophoresis on an agarose gel, blotted onto a membrane, and the membrane probed with a labeled probe specific to the Fragile X locus. This method utilizes the size separation capability of electrophoresis to measure the size of the repeat region, and is labor intensive, time consuming, and requires subjective interpretation of fragment size.
Other published methods for determining the length of a trinucleotide repeat region involve amplifying the target region by PCR followed by size evaluation by capillary electrophoresis using a sequencing instrument. PCR of the target region is performed using primers that straddle the repeat region. The PCR has been optimized to amplify up to 1,000 or more repeats with the best of these methods.
Interpreting electrophoresis results on a conventional planar gel can be challenging. The size reading is somewhat subjective and involves comparing excursion distances between a standard and a sample, assuming insignificant distortion across the gel. One PCR-gel method utilizes repeat primers, in which the primers are full or partial complements of the repeat motif of the target. Electrophoresis of PCR products made with repeat primers results in a smear; the products are a mixture of many different products of different lengths. Interpreting these repeat-primer PCR electrophoresis images is subjective.
Another published method utilizes repeat primers as an alternate to the straddle primers, with high-resolution evaluation on a capillary sequencing instrument. While resolution and clarity of results are improved vs. the planar electrophoresis interpretation can be challenging, particularly in cases of PCR stutter.
All of the methods that utilize capillary sequencing instruments as the reading mechanism are limited by the high cost of those instruments, and by the fact that their operation (such as ambient temperature range) and maintenance requirements are quite stringent.